Validating miR-181a-5p Binding to PTEN 3'-UTR

The direct binding of miR-181a-5p to the 3′-UTR of PTEN was validated by the luciferase reporter assay as described previously [53 (link), 54 (link)]. In brief, the wild-type (WT) PTEN 3′-UTR with position 2310-2316 or truncated (TNC) PTEN 3′-UTR without position 2310-2316 was cloned into the sites 3′ of the firefly luciferase gene (luc2) of pmirGLO luciferase reporter plasmids (Promega Corporation, Madison, WI, USA), which were then cotransfected with miR-181a-5p mimic or mimic-NC to the HEK293T cells using Lipofectamine 6000 reagent (Beyotime). 48 h later, the fluorescent changes were measured using the dual luciferase reporter assay system with the firefly luciferase activity normalized to the corresponding Renilla luciferase activity.